PROJECT SUMMARY: Current antiretrovirals control HIV infections but do not cure the disease. An underdeveloped area in anti-HIV drug discovery is to revitalize and harness the power of the immune system to combat and even eliminate HIV. The HIV-1 Nef protein modulates host immunity to promote disease progression to AIDS. Two of the most sig- nificant functions of Nef are surface downregulation of CD4 and surface downregulation of class I major histo- compatibility complex (MHC-I). Nef?s action on CD4 ensures proper processing of an important component of the virus, the viral Env protein. This action also helps the virus conceal epitopes and escape detection by anti- bodies, thereby avoiding antibody-dependent cellular cytotoxicity (ADCC) that is mediated by the host?s natural killer cells for clearing the infection. In addition, Nef disrupts another essential immune recognition mechanism by downregulating MHC-I. Nef?s action here blocks antigen presentation, which is mediated by MHC-I at the surface of the infected cells. Consequently, due to the lack of viral antigen on the cell surface, infections are hidden from immune surveillance and thus successfully evade the killing by the cytotoxic T cells. Given Nef?s abilities to block these key immune mechanisms, it is conceivable that inhibitors of Nef may reverse such ma- nipulation, which should enable detection and possibly clearance of the infection by the host immune system. Convinced by this hypothesis, we aim to develop antiretrovirals through Nef inhibition, which is our long-term goal. In this project, we are inspired by our recent structural findings that Nef uses a common binding site to execute both functions described above. This is exciting because we could potentially use a single drug to block both essential functions, which may restore tremendous immune power to fight and even eliminate HIV. We propose to use a paralleled approach to identify inhibitors capable of binding to this multifunctional site on Nef. Uniquely, we have designed our drug screening experiments in a way that Nef can adopt biologically ac- tive conformations due to the presence of its coopted cellular partners, namely clathrin AP complexes. Our ap- proach is innovative because our design ensures proper formation of the substrate-binding pockets on Nef that involve the targeted multifunctional site. Our specific aims are: 1) identify inhibitors that block CD4 downregula- tion by Nef. Here we will use both an in vitro fluorescence polarization-based, high throughput screening (HTS) assay and a cell-based CD4 downregulation assay to search for Nef inhibitors that block CD4 binding and downregulation; 2) identify inhibitors that block MHC-I downregulation by Nef. Here we will use a similar fluo- rescence polarization-based HTS assay for screening inhibitors of Nef that can disrupt the binding of MHC-ICD to Nef and clathrin AP1. Successful completion of this work will yield dual-functional inhibitors of Nef, which may serve as drug candidates to be further developed into novel, real world antiretrovirals with unparalleled therapeutic potentials.